Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/834—Urine; urinary system

Definitions

• This invention relates to a method for producing a human epidermal growth factor.
• a human epidermal growth factor is contained in human urine.
• This substance is a linear polypeptide with a molecular weight of about 6,000 and 3 sets of disulfide cross-links between molecules, bearing a close resemblance with mouse epidermal growth factor as obtained from the mouse submandibular gland in chemical structure and in physiological activity.
• This compound has such properties as gastric acid secretion inhibiting activity, epidermal growth promoting activity, etc. and are, therefore, of value as a medicine.
• the present inventors conducted extensive research for developing a method which would be free from the disadvantages of the conventional methods and with which the desired factor could be easily isolated at low cost by a mass treatment of urine.
• the present invention encompasses a method of obtaining a human epidermal growth factor which comprises contacting particles of an aluminium or magnesium silicate of the formula: wherein Z is AL 2 0 3 or 2MgO, v is 2 or 6, and x is the number of water molecules in the compound as is determined by the water content of the silicate compound, with human urine under neutral to acid conditions to thereby absorb said factor on said particles, and eluting said factor with an alkaline solution.
• human epidermal growth factor is selectively adsorbed on adsorbents having the formulas Al 2 O 3 .2SiO 2 ,2H 2 O, Al 2 O 3 -eSiO 2 - 2H 2 O and 2MgO ⁇ 6SiO 2 ⁇ xH 2 O but is not adsorbed at all on adsorbents of the formulas Al 2 O 3 ⁇ 9SiO 2 ⁇ xH 2 O, ySiO 2 ⁇ xH 2 O and Al(OH) 3 , wherein x is the number of water molecules in the compound, as is determined by the water content of the silicate compound.
• the pertinent experimental data are given in Table 1.
• the adsorbents were those selected in consideration of practical utility, i.e. low cost, availability, regeneratability, suitability for the treatment of large volumes of urine, and compatibility with both a batch process and a column process.
• human epidermal growth factor was adsorbed from human urine on various adsorbents under various pH and eluted with IN-aqueous ammonia and the rate of recovery of human epidermal growth factor from urine and the purity thereof were investigated.
• human epidermal growth factor is substantially not adsorbed on adsorbents No. 4 (A!(OH) 3 ), No. 11 (Al 2 O 3 ⁇ 9SiO 2 .xH 2 O) and No. 12 (ySiO 2 .xH 2 O).
• Urine contains a variety of physiologically active substances such as plasminogen activator, uropepsin, and other proteases, and the urine of a pregnant woman contains placental gonadotropic hormone as well. These physiologically active substances should not be contained in the product human epidermal growth factor.
• test urines both normal human urine and pregnant urine were employed.
• this invention relates to a method for producing a human epidermal growth factor which comprises adding particles of an aluminium or magnesium silicate of the formula (wherein Z is A1 2 0 3 or 2MgO; x is the number of water molecules in each molecule of the compound as governed by the water content of the compound) to human urine under neutral to acidic conditions to thereby adsorb the active factor in the urine on said particles and, then, eluting said active factor with an alkaline solution.
• an aluminium or magnesium silicate of the formula wherein Z is A1 2 0 3 or 2MgO; x is the number of water molecules in each molecule of the compound as governed by the water content of the compound
• urine as such may be used in the practice of this invention, it is preferable to pre-adjust urine to pH 8 to 9, preferably pH 8.5, so as to precipitate impurities such as mucopolysaccharides and remove the impurities by a suitable procedure such as filtration.
• the adsorbents used for adsorbing the human epidermal growth factor contained in urine are represented by the formula A1 1 0 3 -2(or 6)SiO 2 ⁇ xH 2 O or 2MgO ⁇ 6SiO ⁇ xH2O.
• the former may be mentioned aluminosilicates of the formula Al 2 O 3 .6SiO 3 .2H 2 0 (adsorbent No. 1 in Table 1) and the formula Al 2 O 3 .2SiO 2 .2H 2 0 (adsorbent No. 2 in Table 1).
• magnesium silicates of the formula 2MgO ⁇ 6SiO 2 ⁇ xH 2 O (adsorbent No. 10 in Table 1).
• adsorbents are added to urine under neutral to acidic conditions.
• the adsorbent is an aluminiumsilicate as defined hereinbefore
• the preferred pH range is 5 to 7. At any pH below 5, impurities tend to be adsorbed.
• the contact thereof with urine is preferably effected batchwise. For practical purposes, it is preferable to contact 2.5 to 5 grams of the adsorbent with each 1 liter of urine, although the ratio of the adsorbent to urine and the contact time are not limited to these specific figures.
• the particle diameter of the adsorbent is not especially critical insofar as the object of this invention can be accomplished.
• the particle size range of 0,149 to 0,074 mm is desirable for a batch process, while the range of 0,30 to 0,149 mm (50 to 100 meshes) is desirable for columnwise adsorption.
• the adsorbent is preferably added to urine under acidic conditions, preferably at pH 2 to 3. At pH values over 3, the adsorption of human epidermal growth factor tends to be inhibited. While the amount of the adsorbent is not especially critical, it is practically desirable to employ 5 to 10 grams for each 1 liter of urine. (All references to mesh sizes herein are Tyler Standard Mesh).
• the adsorbent on which the desired factor has been adsorbed is for example, allowed to settle and separated from the supernatant fluid.
• the factor is then eluted with an alkaline solution, preferably at pH 10 to 12.
• This solution may for example be 1 to 2 N-aqueous ammonia or a 1 to 2 N-aqueous solution of sodium carbonate.
• an alkaline solution preferably at pH 10 to 12.
• This solution may for example be 1 to 2 N-aqueous ammonia or a 1 to 2 N-aqueous solution of sodium carbonate.
• To concentrate the factor in the eluate there may be employed any of the various processes such as the process comprising adding a water-miscible organic solvent such as acetone or a salt such as ammonium sulfate to the eluate to thereby precipitate the factor or the process in which water is removed from the eluate by freeze-drying or vacuum distillation.
• the salting-out process comprising addition of ammonium sulfate
• Human urine which is employed in the practice of this invention may be one which has been preliminarily purified with a certain adsorbent so as to remove impurities.
• adsorbent is an aluminiumsilicate of the formula Al 2 0 3' 9SiO 2 .xH 2 0 (wherein x is as defined hereinbefore), which is available under the tradename of KYOWAAD O 700 (Kyowa Chemical Industries, Ltd.) This can be used for adsorbing impurities in the urine by whichever of a batch process and a columnwise process.
• the adsorption is preferably conducted under weakly acidic conditions, preferably at pH 3 to 5. At pH levels over 5, the efficiency of adsorption of impurities tends to be reduced.
• the preferred particle diameter of the adsorbent is 0,30 to 0,149 mm (50 to 200 meshes), although sizes outside the above range may be used only if the adsorbent is particulate.
• the amount of the adsorbent is not especially critical but the range of 5 to 10 g per liter of urine is preferred.
• the time of contact with urine is generally about 2 hours at the maximum.
• adsorbent used for adsorbing impurities are those materials which are generally represented by the formula Y Si0 2 -xH 2 0, such as diatomaceus earth, and silica gel.
• adsorption is effected under weakly acidic conditions, preferably at pH 4 to 6.
• the adsorption procedure, preferred particle size, amount and contact time, with these adsorbent are the same as those stated for the aluminosilicate.
• aluminum hydroxide is also employed. This material is generally represented by the formula Al(OH) 3 .
• adsorption is preferably conducted under substantially neutral conditions and the preferred amount of the adsorbent is 2.5 to 5 grams per liter of urine.
• the adsorption orocedure. D referred particle size, and contact time are the same as those stated for said aluminiumsilicate.
• any of the above-mentioned aluminiumsilicate, silicic acid and aluminum hydroxide can be used as an adsorbent for adsorbing and removing impurities preliminarily from the urine.
• these adsorbents may similarly be employed but the aluminiumsilicate is particularly useful.
• the eluate was adjusted to pH 5.6 and ammonia sulfate was added to 50% saturation, whereby human epidermal growth factor was precipitated.
• the yield of human epidermal growth factor was 28.5 ug, the purity thereof relative to total protein was 0.25%, and the percent yield was 95%.
• the adsorbent was washed with 10 ml of water and elution was carried out with 20 ml of 1 N-aqueous ammonia. After the eluate was adjusted to pH 5.6, ammonium sulfate was added to 50% saturation, whereby human epidermal growth factor was precipitated.
• the amount of human epidermal growth factor thus obtained was 26.8 pg, the purity thereof based on total protein was 0.43%, and the rate of recovery was 93%.
• the mixture was allowed to stand and the supernataht was separated from the adsorbent.
• the adsorbent was washed with 10 ml of water and elution was carried out with 20 ml of 1 N-aqueous ammonia.
• the eluate was made neutral and ammonium sulfate was added to 50% saturation, whereby human epidermal growth factor was precipitated.
• the amount of human epidermal growth factor thus obtained was 26.5 ug, the purity thereof base on total protein was 0.41%, and the rate of recovery was 88.3%. No placental gonadotropic hormone activity was detected in 1 mg of the protein.
• the determination of human epidermal growth factor activity was carried out in accordance with the radioreceptorassay method of S. Cohen, G. Carpenter: Proc. Nat. Acad. Sci., USA 72, 1317 (1975).
• the standard m-EGF and 125 I-m-EGF used in the assay were prepared by the method of Carpenter et al. (G. Carpenter, S. Cohen et al.: J. Biol. Chem. 250, 4297 (1975).
• protease activity was carried out in accordance with the method of McDonald [C. E. McDonald and L. L. Chen: Biochem. 10, 175 (1965)].
• Human epidermal growth factor prepared according to the method of the invention may be formulated into a pharmaceutical composition, optionally together with one or more pharmaceutically acceptable filters or diluents.

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• Health & Medical Sciences (AREA)
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• Organic Chemistry (AREA)
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• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Toxicology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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• 1982
  • 1982-06-15 JP JP57103292A patent/JPS58219124A/ja active Granted
• 1983
  • 1983-06-09 US US06/502,661 patent/US4528186A/en not_active Expired - Lifetime
  • 1983-06-14 EP EP83303439A patent/EP0097057B1/en not_active Expired
  • 1983-06-14 DE DE8383303439T patent/DE3379379D1/de not_active Expired

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